Clostridium perfringens enterotoxin (CPE) has now been conclusively identified as the virulence factor responsible for symptoms associated with several of the most common foodborne and nonfoodborne gastrointestinal (GI) illnesses of bacterial origin. The long term objective of this project is to fully understand the pathogenesis of CPE-associated GI diseases, including identification of the mechanism of action of CPE, and to identify strategies to prevent or treat these illnesses. To progress towards this goal, the following specific aims will be pursued in the next grant period: 1) evaluating the importance of claudins as CPE receptors for human intestinal cells through Northern analyses and "anti-receptor" antibody studies; if claudins are confirmed as important CPE receptors, claudin: CPE interactions will be explored by phenotyping a series of claudin random mutants for their ability to bind CPE and convey cytotoxicity, 2) identifying the eucaryotic protein constituents of CPE-containing small and large complexes by immunoblot and immunoprecipitation analyses; the importance of each eucaryotic complex protein for CPE action will then be dissected through antibody inhibition studies, 3) using site-directed mutagenesis to high-resolution map CPE functional regions, including the recently identified receptor-binding and large complex-forming regions of the toxin; results generated with these CPE mutants will then be interpreted within the context of the 3-D structure of CPE, and 4) dissecting the molecular pathogenesis of cpe-positive isolates by physical mapping of the cpe plasmid in nonfoodborne disease isolates, determining whether the cpe plasmid can be transferred to other C. perfringens isolates, evaluating whether the chromosomal cpe of food poisoning isolates is dn a mobilizable transposon, and determining whether two component regulatory systems and/or the exponential growth phase repressor Hpr play a role in regulating the sporulation-associated expression of CPE.